Neuroprotective cb2 receptor agonists

ABSTRACT

A method of treating or preventing a neuroinflammatory and/or neurodegenerative disease in a subject by administering a pharmaceutically effective amount of a CB 2  receptor agonist is described. The CB 2  receptor agonist can be a compound according to formula I 
     
       
         
         
             
             
         
       
     
     or a pharmaceutically acceptable salt thereof, with R 1  and R 2  as defined herein. Administration of the CB 2  receptor agonist activates CB 2  receptors in the microglia, and can restore syntaptic plasticity, cognition, and memory in subjects having elevated levels of amyloid-β peptide in the brain.

This application is a continuation of U.S. patent application Ser. No.16/692,098, filed Nov. 22, 2019, which is a continuation of U.S. patentapplication Ser. No. 14/912,647, filed Feb. 18, 2016, which is a 371U.S. National Patent Appln. of PCT/2013/050184, filed Jul. 12, 2013,which claims the benefit of U.S. Provisional Application Ser. No.61/671,277, filed Jul. 13, 2012. The disclosures of each of which isincorporated by reference herein in its entirety.

BACKGROUND

Alzheimer's disease (AD) is an age-dependent neurodegenerative disordercharacterized by progressive loss of memory and cognitive function. Thebrains of patients with AD are characterized by extensive deposits ofextracellular aggregation of amyloid-β (Aβ) peptides. These peptidesform senile plaques and intracellular aggregation of hyperphosphorylatedtau protein. The abnormal accumulation of amyloid fibrils result in theprogressive loss of neuronal circuitry, impairment of the synapticplasticity in the brain, and eventual memory deficiency. In addition,amyloid fibrils activate the inflammatory pathway, characterized by theactivated microglia and astrocytes seen in the brains of patients withAD. It is possible that neuroinflammation could induce beneficial immuneresponses resulting in the phagocytosis of amyloid fibrils in an attemptto limit the development of the disease. Wyss-Coray, T., Nat. Med. 12,1005-1015 (2006). However, prevailing evidence suggests thatneuroinflammation could be a driving force in accelerating AD throughthe production of the proinflammatory chemokines, cytokines, andneurotoxins by the activated microglia and astrocytes in the brain.Perry et al., Nat. Rev. Neurol. 6, 193-201 (2010).

The cannabinoid receptor (CB) family currently includes two clonedmetabotropic receptors: CB₁ (found predominantly in the brain), and CB₂(found primarily in the peripheral immune system) and to a lesser degreein the central nervous system and microglia. In vivo injection of Aβ₁₋₄₂into the hippocampus rat brain resulted in an increase in theendocannabinoid levels (van der Stelt et al., Cell. Mol. Life Sci. 63,1410-1424 (2006)), and anandamide has been shown to prevent Aβ toxicityin cell culture (Milton, Neurosci. Lett. 332, 127-130 (2002)).

With the exception of a small population of neurons located in the brainstem and the cerebellum, healthy brain tissue does not express CB₂receptors. Van Sickle et al., Science 310, 329-332 (2005). Rather, CB₂receptors are upregulated in reactive microglial cells in AD,Huntington's disease, simian immunodeficiency virus-inducedencephalitis, HIV encephalitis, and multiple sclerosis. In vitro studiesdemonstrated that CB₂-selective agonists blocked microglia-mediatedneurotoxicity after Aβ is added to rat cortical co-cultures.Furthermore, intracerebroventricular administration of nonselectivecannabinoid receptor agonist WIN 55,212-2 to rats prevented βP-inducedmicroglial activation, cognitive impairment, and loss of neuronalmarkers. CB₂ agonists are neuroprotective and they have the advantage oflacking the psychotropic adverse effects normally seen with CB₁agonists. Ramirez et al., J. Neurosci. 25, 1904-1913 (2005). Incontrast, CB₁ agonists appear to have no beneficial effects on ADneuropathology and behavioral deficits in a mouse model of AD Chen etal., Curr. Alzheimer Res. 7, 255-261 (2010).

Studies show that in the settings of AD, microglia and astrocytes becomefully reactive, initiating a proinflammatory cascade that results in therelease of potentially neurotoxic substances, including cytokines, whichlead to degenerative changes in neurons. CB₂ is also upregulated duringthis process. The inventors' previous work established1-((3-benzyl-3-methyl-2, 3-dihydro-1-benzofuran-6-yl) carbonyl)piperidine (MDA7) as a novel, bloodbrain barrier-permeant, and highlyselective CB₂ agonist. Diaz et al., Chemmedchem 4, 1615-1629 (2009).MDA7 lacks activity in CB₂knockout mice and its effects in rats andC57BL/6 wild type mice are antagonized by CB₂ antagonists. Naguib etal., Anesth. Analg. 114, 1104-1120 (2012). The neuroprotective effect ofMDA7 was found to be mediated through prevention of glial activation invivo and in in vitro models. The potential effect of MDA7 in otherglial-related pathophysiological conditions such as AD remainsunexplored.

SUMMARY OF THE INVENTION

Cannabinoid type 2 (CB₂) agonists are neuroprotective and appear to playmodulatory roles in neurodegenerative processes in Alzheimer's disease.The inventors studied the effect of MDA7—a novel selective CB₂ agonistthat lacks psychoactivity—on ameliorating the neuroinflammatory process,synaptic dysfunction, and cognitive impairment induced by bilateralmicroinjection of amyloid-beta (Aβ₁₋₄₀) fibrils into the hippocampal CA1area of rats. In rats injected with Aβ₁₋₄₀ fibrils, compared to theadministration of intraperitoneal (i.p.) saline for 14 days, treatmentwith 15 mg/kg of MDA7 i.p. daily for 14 days (i) ameliorated theexpression of CD11b (microglia marker) and GFAP (astrocyte marker), (ii)decreased the secretion of IL-1β, (iii) decreased the upsurge of CB₂receptors, (iv) promoted Aβ clearance, and (v) restored synapticplasticity, cognition and memory. The findings indicate that MDA7 is aninnovative therapeutic approach for the treatment of Alzheimer'sdisease.

In one aspect, the invention provides a method of treating or preventinga neuroinflammatory and/or neurodegenerative disease in a subject byadministering a pharmaceutically effective amount of a CB₂ receptoragonist to the subject. In some embodiments, the CB₂ receptor agonistcomprises a compound according to Formula I:

or a pharmaceutically acceptable salt thereof, wherein: R¹ is selectedfrom the group consisting of cycloalkyl or heterocycloalkyl, any carbonatom of which may be optionally substituted; and R² is selected from thegroup consisting of aryl, cycloalkyl, aralkyl, and alkenyl, any carbonatom of which may be optionally substituted.

Another aspect of the invention provides a method of activating CB₂receptors in microglia of a subject by administering a pharmaceuticallyeffective amount of a CB₂ receptor agonist to the subject. In someembodiments, the subject has elevated levels of amyloid-β peptide in thebrain. In other embodiments, activating the CB₂ receptors increases oneor more of synaptic plasticity, cognition, or memory of the subject. Infurther embodiments, activating the CB₂ receptors decreases productionof IL-1β by the microglia and inhibits MAPK. In a yet furtherembodiment, activating the CB₂ receptors increases glutamatergicneurotransmission in the subject.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides bar graphs showing that administration of MDA7attenuated amyloid fibril-impaired performance in the Morris water mazetest. (A) MDA7 treatment decreases escape latency scores in the Morriswater maze test in a dose-dependent manner. Animals injected withamyloid (Aβ) Aβ₁₋₄₀ fibrils and treated with MDA7 15 mg/kgintraperitoneally (i.p.) for 14 days had a significantly (P<0.05)shorter escape latency in the Morris water maze test than the animalsinjected with Aβ₁₋₄₀ fibrils and treated with saline i.p. for 14 days atdays 3 and 5. In contrast, MDA7 1.5 mg/kg i.p. for 14 days did notresult in any significant memory enhancement following Aβ₁₋₄₀ fibrilsadministration, however, the effect of the 5 mg/kg MDA7 dose was limitedonly to days 3 and 5. (B) The probe trial was performed on day 6 todetermine the time spent in the target quadrant (TQ or platformquadrant) compared with right quadrant (RQ), opposite quadrant (OQ), andleft quadrant (LQ). Rats injected with Aβ₁₋₄₀ fibrils and treated withMDA7 i.p. 15 mg/kg for 14 days spent the longest time in the TQ thananimals injected with Aβ₁₋₄₀ and treated with saline i.p. for 14 days(P<0.05). (C) Rats injected with Aβ₁₋₄₀ fibrils and treated with salinei.p. for 14 days had a significantly extended escape latency in theMorris water maze test compared with that of the rats that receivedbilateral intracerebral microinjection of artificial cerebrospinal fluidand treated with saline i.p. (controls) or animals injected with Aβ₁₋₄₀and treated with 15 mg/kg MDA7 i.p. for 14 days at days 3 to 5.Administration of AM630 prior to MDA7 treatment abrogated the observedeffects of MDA7 indicating that the effects of MDA7 are mediated bystimulating cannabinoid (CB)₂ receptors. Furthermore, rats injected withAβ₁₋₄₀ and received 5 mg/kg of AM630 i.p. for 14 days alone hadcognitive impairment similar to those animals injected with Aβ₁₋₄₀ andtreated with either saline i.p. or 5 mg/kg AM630 plus 15 mg/kg MDA7 i.p.for 14 days. The rats that received bilateral intracerebralmicroinjection of artificial cerebrospinal fluid and treated with salinei.p. or 15 mg/kg MDA7 for 14 days showed no significant difference intheir escape latency values. (D) During the probe trial, the ratsinjected with Aβ₁₋₄₀ fibrils and treated with MDA7 15 mg/kg i.p. for 14days spent the longest time in the TQ than animals injected with Aβ₁₋₄₀and treated for 14 days with either saline i.p., AM630 alone, or AM630plus MDA7 (P<0.01). Statistical significance was determined by repeatedmeasures analysis of variance followed by Student Newman-Keuls multiplerange test.

FIG. 2 provides graphs and images showing that administration of MDA7attenuated amyloid fibrils-induced CD11b immunoreactivity in thehippocampal CA1 area. (A) Coronal rat brain section showing hippocampalink injection: India ink was injected to confirm that the injectionprocedure was accurate and that the material was injected specificallyinto the hippocampal CA1 area of the rat. (B) Immunofluorescence imagesof CD11b immunoreactivity in various groups of rats in the hippocampalCA1 areas. (C) Analysis of CD11b immunofluorescence intensity (20sections from 5 animals per group) showed that the amyloid-β(Aβ)₁₋₄₀-injected rats receiving MDA7 treatment intraperitoneally (i.p.)for 14 days had significantly less (P<0.01) immunofluorescence intensitythan did the animals injected with Aβ₁₋₄₀ and treated with saline i.p.for 14 days. (D) Immunoblots of CD11b reactivity in hippocampus CA1areas. (E) Analysis of CD11b immunoreactivity revealed significantincreases in CD11b (P<0.01) intensities in animals injected with Aβ₁₋₄₀and treated with saline i.p. for 14 days compared with that in thecontrol group (n=5-7 per group). These changes were significantly(P<0.01) attenuated in the Aβ₁₋₄₀-injected rats receiving MDA7 treatmenti.p. for 14 days. Note that treatment with MDA7 alone had no significanteffect on the CD11B expression compared with the control rats.Statistical significance was determined by 1-way analysis of variancefollowed by Student-Newman-Keuls multiple range test.

FIG. 3 provides graphs and images showing that administration of MDA7attenuated amyloid fibrils-induced glial fibrillary acidic protein(GFAP) immunoreactivity in the hippocampal CA1 area. (A)Immunofluorescence images of GFAP immunoreactivity in various groups ofrats in the hippocampal CA1 areas. (B) Analysis of GFAPimmunofluorescence intensity (20 sections from 5 animals per group)showed that the amyloid-β (Aβ)₁₋₄₀-injected rats receiving 15 mg/kg MDA7treatment intraperitoneally (i.p.) for 14 days had significantly less(P<0.01) immunofluorescence intensity than did the animals injected withAβ₁₋₄₀ and treated with saline i.p. for 14 days. (C) Immunoblots of GFAPreactivity in the hippocampus CA1 areas. (D) Analysis of GFAPimmunoreactivity revealed significant increases in GFAP (P<0.01)intensities in the rats injected with Aβ₁₋₄₀ and treated with salinei.p. for 14 days compared with those in the control group (n=5-7 pergroup). These changes were significantly (P<0.01) attenuated in theAβ₁₋₄₀-injected rats receiving MDA7 treatment i.p. for 14 days. Notethat treatment with 15 mg/kg MDA7 i.p. alone had no significant effecton the GFAP expression compared with control rats. Statisticalsignificance was determined by 1-way analysis of variance followed byStudent-Newman-Keuls multiple range test.

FIG. 4 provides graphs and images showing that administration of MDA7attenuated Aβ₁₋₄₀ fibrils upregulated cannabinoid (CB)₂ receptorexpression in the hippocampal CA1 area. (A) CB₂ staining andlocalization in the in the hippocampal CA1 area. Immunofluorescenceimages of CB₂ and CD11b in microglia and the coregionalization of CB₂and microglia are shown. (B) No significant CB₂ expression was observedin the control group, but marked CB₂ expression was observed in the ratsinjected with Aβ₁₋₄₀ and treated with saline intraperitoneally (i.p.)for 14 days (P<0.01 vs. control) (n=20 sections from 5 animals pergroup). CB₂ expression was significantly decreased in theAβ₁₋₄₀-injected rats receiving 15 mg/kg MDA7 treatment i.p. for 14 days.(C) Representative immunoblotting bands to show the expression of CB₂receptor in hippocampal CA1 area in all groups; (D) plotted analysis ofCB₂ immunoreactivity in hippocampus CA1 tissues in all groups. Note thatMDA7 treatment significantly reversed the upsurge of CB₂ expressioninduced by Aβ₁₋₄₀ (P<0.01); n=6 per group. Statistical significance wasdetermined by 1-way analysis of variance followed byStudent-Newman-Keuls multiple range test. Data are shown asmean±standard error of the mean. Scale bar=40 μm.

FIG. 5 provides graphs and images showing that administration of MDA7attenuated amyloid-β (Aβ)₁₋₄₀ fibrils upregulated interleukin (IL)-1βlevel in the hippocampal CA1 area. (A) Immunoblots of IL-1β reactivityin hippocampus CA1 tissues in all groups; (B) plotted analysis of IL-1βimmunoreactivity in hippocampus CA1 tissues in all groups. Note that 15mg/kg MDA7 treatment intraperitoneally (i.p.) for 14 days significantlyreversed the increase of IL-1β protein expression induced by (P<0.01;n=5 per group; 1-way analysis of variance followed byStudent-Newman-Keuls multiple range test). Data are shown asmean±standard error of the mean.

FIG. 6 provides graphs and images showing that the cannabinoid (CB)₂agonist MDA7 induces amyloid-β (Aβ)₁₋₄₀ clearance. (A)Immunofluorescence images of Aβ₁₋₄₀ deposits in the hippocampal CA1areas in different groups. (B) Rats injected with Aβ₁₋₄₀ and treatedwith 15 mg/kg MDA7 intraperitoneally (i.p.) for 14 days exhibited areduction of the deposited Aβ peptide (P<0.01 vs. the rats injected withAβ₁₋₄₀ and treated with saline i.p. for 14 days; n=20 sections from 5animals per group) and this effect was abrogated by prior administrationof 5 mg/kg AM630 i.p. for 14 days. Administration of 5 mg/kg AM630 i.p.alone has no effect on the clearance of the injected Aβ₁₋₄₀. Statisticalsignificance was determined by 1-way analysis of variance followed byStudent-Newman-Keuls multiple range test. Data are shown asmean±standard error of the mean. Scale bar=80 μm.

FIG. 7 provides graphs showing that systemic administration of 15 mg/kgMDA7 intraperitoneally (i.p.) for 14 days ameliorated amyloid-β(Aβ)₁₋₄₀-impaired basal glutamatergic strength and long-termpotentiation (LTP) in the hippocampal CA1 slices. LTP was induced by theelectric stimuli on the Schaffer collateral commissural fibers at 100 Hzfor 1 second. (A) Representative traces of evoked excitatorypostsynaptic currents (EPSCs) at 3 graded stimulus intensities in allgroups. (B) Input (stimulus intensity)-output (EPSC current) curve ofevoked EPSCs in the CA1 neurons in all 4 groups (n=9-10 neurons pergroup). (C) Representative traces to show the evoked EPSCs at baseline,30, and 60 minutes after electric induction in all 4 groups. (D) Timecourse of the LTP induction in the hippocampal CA1 neurons in all 4groups (n=9-12 neurons from 4 to 5 rats per group). Statisticalsignificance was determined by repeated measures analysis of variancefollowed by Student-Newman-Keuls multiple range test. Data are shown asmean±SEM. *P<0.05; **P<0.01 versus Aβ₁₋₄₀ and Aβ₁₋₄₀+MDA7 groups.

DETAILED DESCRIPTION OF THE INVENTION

The inventors have demonstrated that activation of central microglialCB2 receptors by MDA7 promotes Aβ clearance, ameliorates Aβ-inducedglial activation and production of IL-1β, and restores synapticplasticity, cognition and memory. Accordingly, the present inventionprovides CB2 receptor agonists that can be used for the treatment ofneuroinflammatory and/or neurodegenerative diseases including asAlzheimer's disease.

Definitions

As used herein, the terms below have the meanings indicated. As used inthe description of the invention and the appended claims, the singularforms “a”, “an”, and “the” are inclusive of their plural forms, unlesscontraindicated by the context surrounding such.

The term “alkenyl,” as used herein, alone or in combination, refers to astraight-chain or branched-chain hydrocarbon radical having one or moredouble bonds and containing from 2 to 20, preferably 2 to 6, carbonatoms. Alkenylene refers to a carbon-carbon double bond system attachedat two or more positions such as ethenylene [(CH═CH—), (—C::C—)].Examples of suitable alkenyl radicals include ethenyl, propenyl,2-methylpropenyl, 1,4-butadienyl and the like.

The term “alkoxy,” as used herein, alone or in combination, refers to analkyl ether radical, wherein the term alkyl is as defined below.Examples of suitable alkyl ether radicals include methoxy, ethoxy,n-propoxy, isopropoxy, n-butoxy, iso-butoxy, sec-butoxy, tert-butoxy,and the like.

The term “alkyl,” as used herein, alone or in combination, refers to astraight-chain or branched-chain alkyl radical containing from 1 to andincluding 20, preferably 1 to 10, and more preferably 1 to 6, carbonatoms. Alkyl groups may be optionally substituted as defined herein.Examples of alkyl radicals include methyl, ethyl, n-propyl, isopropyl,n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, iso-amyl, hexyl,octyl, noyl and the like. The term “alkylene,” as used herein, alone orin combination, refers to a saturated aliphatic group derived from astraight or branched chain saturated hydrocarbon attached at two or morepositions, such as methylene (—CH₂—).

The term “alkynyl,” as used herein, alone or in combination, refers to astraight-chain or branched chain hydrocarbon radical having one or moretriple bonds and containing from 2 to 20, preferably from 2 to 6, morepreferably from 2 to 4, carbon atoms. “Alkynylene” refers to acarbon-carbon triple bond attached at two positions such as ethynylene(—C:::C—, —C≡C—). Examples of alkynyl radicals include ethynyl,propynyl, hydroxypropynyl, butyn-1-yl, butyn-2-yl, pentyn-1-yl,3-methylbutyn-1-yl, hexyn-2-yl, and the like.

The term “amino,” as used herein, alone or in combination, refers to-NRR′, wherein R and R′ are independently selected from the groupconsisting of hydrogen, alkyl, acyl, heteroalkyl, aryl, cycloalkyl,heteroaryl, and heterocycloalkyl, any of which may themselves beoptionally substituted.

The term “aryl,” as used herein, alone or in combination, means acarbocyclic aromatic system containing one, two or three rings whereinsuch rings may be attached together in a pendent manner or may be fused.The term “aryl” embraces aromatic radicals such as benzyl, phenyl,naphthyl, anthracenyl, phenanthryl, indanyl, indenyl, annulenyl,azulenyl, tetrahydronaphthyl, and biphenyl.

The term “arylalkyl” or “aralkyl,” as used herein, alone or incombination, refers to an aryl group attached to the parent molecularmoiety through an alkyl group.

The term “heteroaryl” means an aryl radical interrupted with one or morehetero atoms, such as a thiophenyl, thiazolyl or imidazolyl radical,optionally substituted with at least one halogen, an alkyl containingfrom 1 to 3 carbon atoms, an alkoxyl, an aryl radical, a nitro function,a polyether radical, a heteroaryl radical, a benzoyl radical, an alkylester group, a carboxylic acid, a hydroxyl optionally protected with anacetyl or benzoyl group, or an amino function optionally protected withan acetyl or benzoyl group or optionally substituted with at least onealkyl containing from 1 to 6 carbon atoms.

The term “cycloalkyl,” or, alternatively, “carbocycle,” as used herein,alone or in combination, refers to a saturated or partially saturatedmonocyclic, bicyclic or tricyclic alkyl radical wherein each cyclicmoiety contains from 3 to 12, preferably five to seven, carbon atom ringmembers and which may optionally be a benzo fused ring system which isoptionally substituted as defined herein. Examples of such cycloalkylradicals include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl, octahydronaphthyl, 2,3-dihydro-1H-indenyl, adamantyl andthe like. “Bicyclic” and “tricyclic” as used herein are intended toinclude both fused ring systems, such as decahydronapthalene,octahydronapthalene as well as the multicyclic (multicentered) saturatedor partially unsaturated type. The latter type of isomer is exemplifiedin general by, bicyclo[1,1, 1]pentane, camphor, adamantane, andbicyclo[3,2,1]octane.

The term “ester,” as used herein, alone or in combination, refers to acarboxy group bridging two moieties linked at carbon atoms.

The term “ether,” as used herein, alone or in combination, refers to anoxy group bridging two moieties linked at carbon atoms.

The term “halo,” or “halogen,” as used herein, alone or in combination,refers to fluorine, chlorine, bromine, or iodine.

The term “haloalkyl,” as used herein, alone or in combination, refers toan alkyl radical having the meaning as defined above wherein one or morehydrogens are replaced with a halogen. Specifically embraced aremonohaloalkyl, dihaloalkyl and polyhaloalkyl radicals. A monohaloalkylradical, for one example, may have an iodo, bromo, chloro or fluoro atomwithin the radical. Dihalo and polyhaloalkyl radicals may have two ormore of the same halo atoms or a combination of different halo radicals.Examples of haloalkyl radicals include fluoromethyl, difluoromethyl,trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl,pentafluoroethyl, heptafluoropropyl, difluorochloromethyl,dichlorofluoromethyl, difluoroethyl, difluoropropyl, dichloroethyl anddichloropropyl. “Haloalkylene” refers to a haloalkyl group attached attwo or more positions. Examples include fluoromethylene (—CFH—),difluoromethylene (—CF₂—), chloromethylene (—CHCl—) and the like.

The term “heteroalkyl,” as used herein, alone or in combination, refersto a stable straight or branched chain, or cyclic hydrocarbon radical,or combinations thereof, fully saturated or containing from 1 to 3degrees of unsaturation, consisting of the stated number of carbon atomsand from one to three heteroatoms selected from the group consisting ofO, N, and S, and wherein the nitrogen and sulfur atoms may optionally beoxidized and the nitrogen heteroatom may optionally be quaternized. Theheteroatom(s) O, N and S may be placed at any interior position of theheteroalkyl group. Up to two heteroatoms may be consecutive, such as,for example, —CH₂—NH—OCH₃.

The term “heteroaryl,” as used herein, alone or in combination, refersto 3 to 7 membered, preferably 5 to 7 membered, unsaturatedheteromonocyclic rings, or fused polycyclic rings in which at least oneof the fused rings is unsaturated, wherein at least one atom is selectedfrom the group consisting of O, S, and N. The term also embraces fusedpolycyclic groups wherein heterocyclic radicals are fused with arylradicals, wherein heteroaryl radicals are fused with other heteroarylradicals, or wherein heteroaryl radicals are fused with cycloalkylradicals. Examples of heteroaryl groups include pyrrolyl, pyrrolinyl,imidazolyl, pyrazolyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl,triazolyl, pyranyl, furyl, thienyl, oxazolyl, isoxazolyl, oxadiazolyl,thiazolyl, thiadiazolyl, isothiazolyl, indolyl, isoindolyl, indolizinyl,benzimidazolyl, quinolyl, isoquinolyl, quinoxalinyl, quinazolinyl,indazolyl, benzotriazolyl, benzodioxolyl, benzopyranyl, benzoxazolyl,benzoxadiazolyl, benzothiazolyl, benzothiadiazolyl, benzofuryl,benzothienyl, chromonyl, coumarinyl, benzopyranyl, tetrahydroquinolinyl,tetrazolopyridazinyl, tetrahydroisoquinolinyl, thienopyridinyl,furopyridinyl, pyrrolopyridinyl and the like. Exemplary tricyclicheterocyclic groups include carbazolyl, benzidolyl, phenanthrolinyl,dibenzofuranyl, acridinyl, phenanthridinyl, xanthenyl and the like.

The terms “heterocycloalkyl” and, interchangeably, “heterocyclyl,” asused herein, alone or in combination, each refer to a saturated,partially unsaturated, or fully unsaturated monocyclic, bicyclic, ortricyclic heterocyclic radical containing at least one, preferably 1 to4, and more preferably 1 to 2 heteroatoms as ring members, wherein eachsaid heteroatom may be independently selected from the group consistingof nitrogen, oxygen, and sulfur, and wherein there are preferably 3 to 8ring members in each ring, more preferably 3 to 7 ring members in eachring, and most preferably 5 to 6 ring members in each ring.“Heterocycloalkyl” and “heterocyclyl” are intended to include sulfones,sulfoxides, N-oxides of tertiary nitrogen ring members, and carbocyclicfused and benzo fused ring systems; additionally, both terms alsoinclude systems where a heterocycle ring is fused to an aryl group, asdefined herein, or an additional heterocycle group. Heterocyclyl groupsof the invention are exemplified by aziridinyl, azetidinyl,1,3-benzodioxolyl, dihydroisoindolyl, dihydroisoquinolinyl,dihydrocinnolinyl, dihydrobenzodioxinyl,dihydro[1,3]oxazolo[4,5-b]pyridinyl, benzothiazolyl, dihydroindolyl,dihy-dropyridinyl, 1,3-dioxanyl, 1,4-dioxanyl, 1,3-dioxolanyl,isoindolinyl, morpholinyl, piperazinyl, pyrrolidinyl,tetrahydropyridinyl, piperidinyl, thiomorpholinyl, and the like. Theheterocyclyl groups may be optionally substituted unless specificallyprohibited.

Any definition herein may be used in combination with any otherdefinition to describe a composite structural group. By convention, thetrailing element of any such definition is that which attaches to theparent moiety. For example, the composite group alkylamido wouldrepresent an alkyl group attached to the parent molecule through anamido group, and the term alkoxyalkyl would represent an alkoxy groupattached to the parent molecule through an alkyl group.

The term “optionally substituted” means the anteceding group may besubstituted or unsubstituted. When substituted, the substituents of an“optionally substituted” group may include, without limitation, one ormore substituents independently selected from the following groups or aparticular designated set of groups, alone or in combination: loweralkyl, lower alkenyl, lower alkynyl, lower alkanoyl, lower heteroalkyl,lower heterocycloalkyl, lower haloalkyl, lower haloalkenyl, lowerhaloalkynyl, lower perhaloalkyl, lower perhaloalkoxy, lower cycloalkyl,phenyl, aryl, aryloxy, lower alkoxy, lower haloalkoxy, oxo, loweracyloxy, carbonyl, carboxyl, lower alkylcarbonyl, lower carboxyester,lower carboxamido, cyano, hydrogen, halogen, hydroxy, amino, loweralkylamino, arylamino, amido, nitro, thiol, lower alkylthio, arylthio,lower alkylsulfinyl, lower alkylsulfonyl, arylsulfinyl, arylsulfonyl,arylthio, sulfonate, sulfonic acid, trisubstituted silyl, N₃, SH, SCH₃,C(O)CH₃, CO₂CH₃, CO₂H, pyridinyl, thiophene, furanyl, lower carbamate,and lower urea. Two substituents may be joined together to form a fusedfive-, six-, or seven-membered carbocyclic or heterocyclic ringconsisting of zero to three heteroatoms, for example formingmethylenedioxy or ethylenedioxy. An optionally substituted group may beunsubstituted (e.g., —CH₂CH₃), fully substituted (e.g., —CF₂CF₃),monosubstituted (e.g., —CH₂CH₂F) or substituted at a level anywherein-between fully substituted and monosubstituted (e.g., —CH₂CF₃). Wheresubstituents are recited without qualification as to substitution, bothsubstituted and unsubstituted forms are encompassed. Where a substituentis qualified as “substituted,” the substituted form is specificallyintended. Additionally, different sets of optional substituents to aparticular moiety may be defined as needed; in these cases, the optionalsubstitution will be as defined, often immediately following the phrase,“optionally substituted with.”

The invention is inclusive of the compounds described herein in any oftheir pharmaceutically acceptable forms, including isomers (e.g.,diastereomers and enantiomers), tautomers, salts, solvates, polymorphs,prodrugs, and the like. In particular, if a compound is opticallyactive, the invention specifically includes each of the compound'senantiomers as well as racemic mixtures of the enantiomers. This is trueregardless of whether or not the enantiomers are shown in chemicalformula representing the compounds. For example, if a compound thatincludes a chiral center is shown without any indication ofstereochemistry, it is presumed to represent all possible stereoisomersof the compound. It should be understood that the term “compound”includes any or all of such forms, whether explicitly stated or not(although at times, “salts” are explicitly stated).

“Treat,” “treating”, and “treatment”, etc., as used herein, refer to anyaction providing a benefit to a subject afflicted with a condition ordisease such as Alzheimer' s disease. Treatment can result in completeremission of the neurodegenerative disease, but can also include lessereffects, including improvement in the condition through lessening orsuppression of at least one symptom, delay in progression of thedisease, avoiding the development of additional symptoms in one known tobe afflicted with Alzheimer's disease, etc.

Prevention, as used herein, refers to any action providing a benefit toa subject at risk of being afflicted with a condition or disease such asAlzheimer's disease, including avoidance of the development ofAlzheimer's disease or a decrease of one or more symptoms of the diseaseshould Alzheimer's disease develop. The subject may be at risk due toadvanced age, as a result of family history, and/or from various otherknown risk factors.

“Pharmaceutically acceptable” as used herein means that the compound orcomposition is suitable for administration to a subject for the methodsdescribed herein, without unduly deleterious side effects in light ofthe severity of the disease and necessity of the treatment.

The term “pharmaceutically effective amount” is intended to qualify theamount of each agent which will achieve the goal of decreasing diseaseseverity while avoiding adverse side effects such as those typicallyassociated with alternative therapies. The pharmaceutically effectiveamount may be administered in one or more doses.

The inventors have determined that the CB₂ receptor functions in anegative-feedback loop and that the administration of a CB₂ agonist canpromote clearance of Aβ, ameliorate the neuroinflammatory response toAβ, and prevent Aβ-induced microglial and astrocyte activation, cytokineproduction, loss of synaptic plasticity, and cognitive impairment.Accordingly, one aspect of the invention provides a method of treatingor preventing a neurodegenerative disease in a subject by administeringa pharmaceutically effective amount of a cannabinoid receptor 2 (CB₂)receptor agonist to the subject. Cannabinoid receptors are a class ofcell membrane receptors under the G protein-coupled receptorsuperfamily, and include subtypes CB₁ and CB₂. A CB₂ receptor agonist,as defined herein, is a compound that exhibits an EC₅₀ or IC₅₀ withrespect to a CB₂ receptor activity of no more than about 100 μM and moretypically not more than about 50 μM, as measured in the cannabinoidreceptor assay described generally herein below. “EC₅₀” is thatconcentration of modulator which activates the activity of a cannabinoidreceptor to half-maximal level. “IC₅₀” is that concentration ofmodulator which reduces the activity of a CB₂ receptor to half-maximallevel.

A neurodegenerative disease is a disease involving progressive loss ofstructure or function of neurons, including death of neurons. There area number of features commonly associated with various types ofneurodegenerative disease, including protein misfolding, proteindegradation pathways, mitochondrial dysfunction, and programmed celldeath. Examples of neurodegenerative diseases include Alzheimer'sdisease, Parkinson's disease, Huntington's disease, and amylotrophiclateral sclerosis. In some embodiments, the disease is aneuroinflammatory disease. Neuroinflammatory diseases involveinflammation of neural tissue, and generally involve the activation ofmicroglia and/or astroglia and the attendant expression ofproinflammatory cytokines and chemokines. The term neuroinflammation hasbeen well defined by those skilled in the art. O′Callaghan et al., Ann.N Y Acad Sci., 1139, 318-30 (2008).

In other embodiments, the disease involves elevated levels of amyloid-(3peptide in the brain of the subject. By “involves,” it is meant that theAP plays a role in the pathology of the disease. Elevated levels referto levels higher than those found by in a healthy subject. There are avariety of ways to determine Aβ levels, including immunostaining, ELISA,and atomic force microscopy. Amyloid beta (Aβ) is a peptide of 36-43amino acids that is processed from the amyloid precursor protein (APP)that is best known as a component of amyloid plaques in association withAlzheimer's disease. Plaques are composed of a tangle of regularlyordered fibrillar aggregates called amyloid fibers. Similar plaquesappear in some variants of Lewy body dementia and in inclusion bodymyositis (a muscle disease), while Aβ can also form the aggregates thatcoat cerebral blood vessels in cerebral amyloid angiopathy.

In some embodiments, CB₂ receptor agonists are used to treat or preventAlzheimer's disease. Alzheimer's disease (AD) is an age-dependentneurodegenerative disorder characterized by progressive loss of memoryand cognitive function. The brains of patients with AD are characterizedby extensive deposits of extracellular aggregation of (Aβ) peptides. Thedisease course is divided into four stages, with progressive patterns ofcognitive and functional impairment, with include pre-dementia, earlyAD, moderate AD, and advanced AD. Symptoms of Alzheimer's diseaseinclude confusion, irritability, aggression, mood swings, trouble withlanguage, and long-term memory loss. When a diagnosis of AD issuspected, the diagnosis can be confirmed by a brain scan and with teststhat evaluate behaviour and thinking abilities. Computed tomography(CT), magnetic resonance imaging (MRI), single-photon emission computedtomography (SPECT), and positron emission tomography (PET) are examplesof imaging technology that can be used to carry out brain scans insubjects suspected of having AD.

A variety of effective CB₂ receptor agonists have been developed. CB₂receptor agonists include non-selective CB₂ receptor agonists andselective CB₂ receptor agonists. Examples of non-selective CB₂ receptoragonists include Δ⁹-tetrahydrocannabinol (Δ⁹-THC),(6αR)-trans-3-(1,1-dimethylheptyl)-6a,7,10,10α-tetrahydro-1-hydroxy-6,6-dimethyl-6H-dibenzo [b,d]pyran-9-methanol (HU-210),(−)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol(CP55940),(R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo-[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone (R-(+)-WIN55212),N-arachidonoyl ethanolamine (anandamide), and 2-arachidonoyl glycerol.

Examples of selective CB₂ receptor agonists include 3-benzyl-3-methyl-2,3-dihydrobenzofuran-6-carboxylic acid-piperidine amide (MDA7),(2-methyl-1-propyl-1H-indol-3-yl)-1-napthalenylmethanone (JWH-015),3-(1,1-dimethylbutyl)-6,6,9-trimethyl-6a,7,10, 10α-tetrahydro-6H-benzo[c]chromene (JWH-133), HU-308,(2-iodo-5-nitrophenyl)-[1-(1-methylpiperidin-2-ylmethyl)-1H-indol-3-yl]-methanone (AM1241), GW405833, GW842166X, andO-1966. The structures and further information on selective andnon-selective CB₂ receptor agonists are described by Guindon et al.(Guindon et al., Brit. J. Pharmacol, 153, 319-334 (2008)) and by RogerPertwee (Pertwee, R, Brit. J. Pharmacol, 156, 397-411 (2009)), thedisclosures of which are incorporated herein by reference. AdditionalCB₂ receptor agonists can be identified through high throughputscreening of compounds developed from known chemical scaffolds, asdescribed by Whiteside et al. (Whiteside et al., Curr Med Chem, 14(8),917-36 (2007)), the disclosure of which is incorporated herein byreference.

Candidate CB₂ receptor agonists may be tested in animal models. Forexample, CB₂ receptor agonists can be tested in rats administeredAβ₁₋₄₀, as described herein. Results are typically compared betweencontrol animals treated with candidate agents and the controllittermates that did not receive treatment. Transgenic animal models arealso available and are commonly accepted as models for human disease(see, for instance, Greenberg et al., Proc. Natl. Acad. Sci. USA,92:3439-3443 (1995)). Candidate agents can be used in these animalmodels to determine if a candidate agent decreases one or more of thesymptoms associated with the Alzheimer's disease, including, forinstance, increased Aβ levels, loss of memory, decreased synapticplasticity, or combinations thereof. For example, hippocampal LTP isused as a correlate for learning and memory and has emerged as avaluable model for studying mechanisms involved in cognitive deficitsrelated to AD

In some embodiments, the CB₂ receptor agonist comprises a compoundaccording to Formula I:

or a pharmaceutically acceptable salt thereof, wherein: R¹ is selectedfrom the group consisting of cycloalkyl or heterocycloalkyl, any carbonatom of which may be optionally substituted; and R² is selected from thegroup consisting of aryl, cycloalkyl, aralkyl, and alkenyl, any carbonatom of which may be optionally substituted. In further embodiments, theCB₂ receptor agonist comprises an S isomer of a compound according toFormula I.

Structure activity studies have been conducted to demonstrate theactivity of CB₂ receptor agonists according to Formula I. See Diaz etal., ChemMedChem 4, 1615-1629 (2009), the disclosure of which isincorporated herein by reference. Some compounds were shown to havehigher activity than others. For example, different activities can beobtained by varying the substituent at R². Accordingly, in someembodiments, R² is an aryl group, while in further embodiments R² is aphenyl group. Different activities can also be obtained by varying R¹.Accordingly, in some embodiments, R¹ is a heterocycloalkyl group, whilein other embodiments, R¹ is a 1-piperidyl group.

In a preferred embodiment, the CB₂ receptor agonist is a compoundaccording to formula II

This compound has the chemical name3-benzyl-3-methyl-2,3-dihydrobenzofuran-6-carboxylic acid-piperidineamide, and is also referred to herein as MDA7.

Additional studies have been carried out to determine if one of theenantiomers of MDA7 is more active than the other. While bothenantiomers are active, the S enantiomer has been shown to displayhigher activity. See Luo et al, Tetrahedron Letters, 53(26), 3316(2012), the disclosure of which is incorporated herein by reference. Thestructure of the S enantiomers ((S)-(3-benzyl-3-methyl-2,3-dihydro-benzofuran-6-yl)-piperidin-l-yl-methanone) is shown in formulaIII below:

In another aspect, a method of activating CB₂ receptors in microglia ofa subject by administering a pharmaceutically effective amount of a CB₂receptor agonist to the subject is provided. The CB₂ receptor agonistsinclude the specific and non-specific CB₂ receptor agonists describedherein.

In some embodiments, the method of activating CB₂ receptors uses a CB₂receptor agonist according to Formula I: In some embodiments, the CB₂receptor agonist comprises a compound according to Formula I:

or a pharmaceutically acceptable salt thereof, wherein: R¹ is selectedfrom the group consisting of cycloalkyl or heterocycloalkyl, any carbonatom of which may be optionally substituted; and R² is selected from thegroup consisting of aryl, cycloalkyl, aralkyl, and alkenyl, any carbonatom of which may be optionally substituted.

In some embodiments, R² is an aryl group, while in further embodimentsR² is a phenyl group. Different activities can also be obtained byvarying R¹. In further embodiments, R¹ is a heterocycloalkyl group,while in other embodiments, R¹ is a 1-piperidyl group.

In a preferred embodiment, the CB₂ receptor agonist is a compoundaccording to formula II

This compound has the chemical name3-benzyl-3-methyl-2,3-dihydrobenzofuran-6-carboxylic acid-piperidineamide, and is also referred to herein as MDA7. In some embodiments, theCB₂ receptor agonist is the S enantiomer((S)-(3-benzyl-3-methyl-2,3-dihydro-benzofuran-6-yl)-piperidin-1-yl-methanone).

In some embodiments of the method of activating CB₂ receptors inmicroglia of a subject, the subject has elevated levels of amyloid-βpeptide in the brain. The inventors have shown that the presence ofelevated levels of amyloid-β peptide in a subject induced the activationof astrocytes and microglia and upregulation of CB₂ receptors in thehippocampal CA1 area, and that treatment with the CB₂ receptor agonistMDA7 reversed this process. Because activation of the microglia resultsin inflammation, suppression of inflammation in neural tissue byreversing microglia activation can be used to treat or preventneurodegerative disease involving neuroinflammation.

The inventors have shown also that the activation of central microglialCB₂ receptors by MDA7 (i) promoted Aβ clearance, (ii) amelioratedAP-induced glial activation and production of IL-1β, and (iii) restoredsynaptic plasticity, cognition and memory. Accordingly, in someembodiments, the method of activating the CB₂ receptors can increase oneor more of synaptic plasticity, cognition, or memory in a subject.Synaptic plasticity is the ability of the connection, or synapse,between two neurons to change in strength in response to either use ordisuse of transmission over synaptic pathways. Since memories arepostulated to be represented by vastly interconnected networks ofsynapses in the brain, synaptic plasticity is one of the importantneurochemical foundations of learning and memory. Synaptic plasticity isgenerally measured as a change in an evoked electrophysiologicalresponse, such as transmitter release or change in relative electricpotential. Definitions and methods of measuring cognition and memorycapability in a subject are well known to those skilled in the art.

In another embodiment, activating the CB₂ receptors decreases productionof IL-1β by the microglia. IL-1β is synthesized and released from theactivated microglia and astrocytes in the brain and is actively involvedin the development of amyloid-induced brain inflammation, impairedglutamatergic transmission and memory deficiency. The inventorsdemonstrated that the levels of interleukin 1β (IL-1β) from activatedglial cells were significantly increased in the hippocampal CA1 areaafter microinjection of Aβ₁₋₄₀, and these increases were blunted by MDA7treatment for 14 days. Decreasing IL-1β is important for at least thereason that IL-1β is an important mediator of neuroinflammation.

In a further embodiment, activating the CB₂ receptors increasesglutamatergic neurotransmission in the subject. Glutamatergicneurotransmission is neurotransmission based on glutamate, which is amajor excitatory neurotransmitter in the mammalian central nervoussystem. Niciu et al., Pharmacol Biochem Behay. 100(4), 656-664 (2012).Glutamatergic neurotransmission involves a variety of ionotropic andmetabotropic receptors, such as NMDA receptors and AMPA/Kainatereceptors. The inventors demonstrated that MDA7 effectively amelioratesglutamatergic transmission that had been impaired by Aβ₁₋₄₀.

The compounds of the invention can be used to provide prophylacticand/or therapeutic treatment. The compounds of the invention can, forexample, be administered prophylactically to a subject in advance of theoccurrence of a neurodegenerative disorder (e.g., Alzheimer's disease).Prophylactic (i.e., preventive) administration is effective to decreasethe likelihood of the subsequent occurrence of neuroinflammatory orneurodegenerative disease in the subject, or decrease the severity of aneuroinflammatory or neurodegenerative disease that subsequently occurs.Prophylactic treatment may be provided to a subject that is at elevatedrisk of developing a neuroinflammatory and/or neurodegenerative disease,such as a subject with a family history of neuroinflammatory orneurodegenerative disease.

The compounds of the invention can also be administered therapeuticallyto a subject that is already afflicted by a neuroinflammatory orneurodegenerative disease. In one embodiment of therapeuticadministration, administration of the compounds is effective toeliminate the neurodegenerative disease; in another embodiment,administration of the compounds is effective to decrease the severity ofthe neuroinflammatory or neurodegenerative disease or lengthen thelifespan of the subject so afflicted. The subject is preferably amammal, such as a domesticated farm animal (e.g., cow, horse, pig) orpet (e.g., dog, cat). More preferably, the subject is a human, in whichcase the subject may also be referred to as a patient.

In some embodiments, the CB₂ receptor agonist may be administered incombination with one or more other agents known to be effective fortreating Alzheimer's disease. By administration in combination with whatis meant is that the compounds are administered to the subject in acontemporaneous manner. Administration in combination does not requirethat the agents be administered simultaneously or that they beformulated together, although in some embodiments this may be the case.Examples of additional agents known to be effective for treatingAlzheimer's disease include acetylcholinesterase inhibitors such astacrine, rivastigmine, galantamine and donepezil and the NMDA receptorantagonist memantine. Administration in combination with an additionalanti-alzheimer's agent is also meant to include administration withsubsequently developed anti-alzheimer's agents.

Administration and Formulation of the Compounds of the Invention

The present invention also provides pharmaceutical compositions thatinclude compounds such as those defined by the formulae described hereinas an active ingredient, and a pharmaceutically acceptable liquid orsolid carrier or carriers, in combination with the active ingredient.Any of the compounds described above as being suitable for the treatmentof cancer can be included in pharmaceutical compositions of theinvention.

The compounds can be administered as pharmaceutically acceptable salts.Pharmaceutically acceptable salt refers to the relatively non-toxic,inorganic and organic acid addition salts of the compounds. These saltscan be prepared in situ during the final isolation and purification ofthe compounds of the invention, or by separately reacting a purifiedcompound of the invention with a suitable counterion, depending on thenature of the compound, and isolating the salt thus formed.Representative counterions include the chloride, bromide, nitrate,ammonium, sulfate, tosylate, phosphate, tartrate, ethylenediamine, andmaleate salts, and the like. See for example Haynes et al., J. Pharm.Sci., 94, p. 2111-2120 (2005).

The pharmaceutical compositions include one or more compounds of theinvention together with one or more of a variety of physiologicalacceptable carriers for delivery to a patient, including a variety ofdiluents or excipients known to those of ordinary skill in the art. Forexample, for parenteral administration, isotonic saline is preferred.For topical administration, a cream, including a carrier such asdimethylsulfoxide (DMSO), or other agents typically found in topicalcreams that do not block or inhibit activity of the compound, can beused. Other suitable carriers include, but are not limited to, alcohol,phosphate buffered saline, and other balanced salt solutions.

The formulations may be conveniently presented in unit dosage form andmay be prepared by any of the methods well known in the art of pharmacy.Preferably, such methods include the step of bringing the active agentinto association with a carrier that constitutes one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing the active agent into association with a liquidcarrier, a finely divided solid carrier, or both, and then, ifnecessary, shaping the product into the desired formulations. Themethods of the invention include administering to a subject, preferablya mammal, and more preferably a human, the composition of the inventionin an amount effective to produce the desired effect. The formulatedcompounds can be administered as a single dose or in multiple doses.Useful dosages of the active agents can be determined by comparing theirin vitro activity and the in vivo activity in animal models. Methods forextrapolation of effective dosages in mice, and other animals, to humansare known in the art; for example, see U.S. Pat. No. 4,938,949.

Formulations for parenteral administration include aqueous andnon-aqueous (oily) sterile injection solutions of the active compoundswhich may contain antioxidants, buffers, bacteriostats and solutes whichrender the formulation isotonic with the blood of the intendedrecipient; and aqueous and non-aqueous sterile suspensions which mayinclude suspending agents and thickening agents. Suitable lipophilicsolvents or vehicles include fatty oils such as sesame oil, or syntheticfatty acid esters, such as ethyl oleate or triglycerides, or liposomes.Aqueous injection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.

One example of a formulation appropriate for administration through aparenteral route comprises 1.00 g of MDA7, 30.00 g of N-methylpyrrolidone, 30.00 g of propylene glycol, 10.00 g of CREMOPHOR® ELP,10.00 g of EtOH (95%), and 19.00 g of saline solution. Another exampleof formulation is based on hydroxypropyl-β-cyclodextrins (HPβCD) asdescribed in Astruc-Diaz, F., McDaniel, S. W., Xu, J J., Parola, S.,Brown, D. L., Naguib, M., Diaz, P. 2013. In vivo efficacy of enablingformulations based on hydroxypropyl-β-cyclodextrins, micellarpreparation, and liposomes for the lipophilic cannabinoid CB₂ agonist,MDA7. J Pharm Sci 102, 352-364.

The agents of the present invention are preferably formulated inpharmaceutical compositions and then, in accordance with the methods ofthe invention, administered to a subject, such as a human patient, in avariety of forms adapted to the chosen route of administration. Theformulations include, but are not limited to, those suitable for oral,rectal, vaginal, topical, nasal, ophthalmic, or parenteral (includingsubcutaneous, intramuscular, intraperitoneal, intratumoral, andintravenous) administration. A preferred form of administration isparenteral administration.

Formulations of the present invention suitable for oral administrationmay be presented as discrete units such as tablets, troches, capsules,lozenges, wafers, or cachets, each containing a predetermined amount ofthe active agent as a powder or granules, as liposomes containing theactive compound, or as a solution or suspension in an aqueous liquor ornon-aqueous liquid such as a syrup, an elixir, an emulsion, or adraught. Such compositions and preparations typically contain at leastabout 0.1 wt-% of the active agent. The amount of the CB₂ receptoragonist (i.e., active agent) is such that the dosage level will beeffective to produce the desired result in the subject.

Nasal spray formulations include purified aqueous solutions of theactive agent with preservative agents and isotonic agents. Suchformulations are preferably adjusted to a pH and isotonic statecompatible with the nasal mucous membranes. Formulations for rectal orvaginal administration may be presented as a suppository with a suitablecarrier such as cocoa butter, or hydrogenated fats or hydrogenated fattycarboxylic acids. Ophthalmic formulations are prepared by a similarmethod to the nasal spray, except that the pH and isotonic factors arepreferably adjusted to match that of the eye. Topical formulationsinclude the active agent dissolved or suspended in one or more mediasuch as mineral oil, petroleum, polyhydroxy alcohols, or other basesused for topical pharmaceutical formulations.

A preferred method for administering a topical pharmaceuticalformulation is a transdermal patch. Transdermal patches dispense a drugat a controlled rate by presenting the drug for absorption in anefficient manner with a minimum of degradation of the drug. Typically,transdermal patches comprise an impermeable backing layer, a singlepressure sensitive adhesive and a removable protective layer with arelease liner. One of ordinary skill in the art will understand andappreciate the techniques appropriate for manufacturing a desiredefficacious transdermal patch based upon the needs of the artisan.

The tablets, troches, pills, capsules, and the like may also contain oneor more of the following: a binder such as gum tragacanth, acacia, cornstarch or gelatin; an excipient such as dicalcium phosphate; adisintegrating agent such as corn starch, potato starch, alginic acid,and the like; a lubricant such as magnesium stearate; a sweetening agentsuch as sucrose, fructose, lactose, or aspartame; and a natural orartificial flavoring agent. When the unit dosage form is a capsule, itmay further contain a liquid carrier, such as a vegetable oil or apolyethylene glycol. Various other materials may be present as coatingsor to otherwise modify the physical form of the solid unit dosage form.For instance, tablets, pills, or capsules may be coated with gelatin,wax, shellac, sugar, and the like. A syrup or elixir may contain one ormore of a sweetening agent, a preservative such as methyl- orpropylparaben, an agent to retard crystallization of the sugar, an agentto increase the solubility of any other ingredient, such as a polyhydricalcohol, for example glycerol or sorbitol, a dye, and flavoring agent.The material used in preparing any unit dosage form is substantiallynontoxic in the amounts employed. The active agent may be incorporatedinto sustained-release preparations and devices.

Preparation of the Compounds

Compounds of the invention may be synthesized by synthetic routes thatinclude processes similar to those well known in the chemical arts. Thestarting materials are generally available from commercial sources suchas Aldrich Chemicals (Milwaukee, Wis., USA) or are readily preparedusing methods well known to those skilled in the art (e.g., prepared bymethods generally described in Louis F. Fieser and Mary Fieser, Reagentsfor Organic Synthesis, v. 1-19, Wiley, N.Y., (1967-1999 ed.); Alan R.Katritsky, Otto Meth-Cohn, Charles W. Rees, Comprehensive OrganicFunctional Group Transformations, v 1-6, Pergamon Press, Oxford,England, (1995); Barry M. Trost and Ian Fleming, Comprehensive OrganicSynthesis, v. 1-8, Pergamon Press, Oxford, England, (1991); orBeilsteins Handbuch der organischen Chemie, 4, Aufl. Ed.Springer-Verlag, Berlin, Germany, including supplements (also availablevia the Beilstein online database)). In particular, methods for thepreparation of a variety of CB₂ receptor agonists are described in U.S.patent application Ser. No. 12/668,840 and U.S. patent application Ser.No. 12/668,867, the disclosures of which are incorporated herein byreference.

The present invention is illustrated by the following examples. It is tobe understood that the particular examples, materials, amounts, andprocedures are to be interpreted broadly in accordance with the scopeand spirit of the invention as set forth herein.

EXAMPLES Example 1: Preparation of3-benzyl-3-methyl-2,3-dihydrobenzofuran-6-carboxylic acid-piperidineamide (MDA7)

A) 4-Hydroxy-3-iodo-benzoic acid: 4-Hydroxybenzoic acid (0.037 mol, 5.1g) was dissolved in 100 mL of methanol. One equivalent each of sodiumiodide (0.037 mol, 5.54 g) and sodium hydroxide (0.037 mol, 1.48 g) wasadded, and the solution was cooled to 0° C. Aqueous sodium hypochlorite(64 ml, 4.0% NaOCl) was added dropwise over 75 min at 0-3° C. As eachdrop hit the solution, a red color appeared and faded almost instantly.The resulting colorless slurry was stirred for 1 h at 0-2° C. and thenwas treated with 40 mL of 10% aqueous sodium thiosulfate. The mixturewas acidified by 4M aqueous HCl. A product crystallized and was filteredoff to afford 1.1 g. Ethyl acetate (250 mL) was added, and the layerswere separated. The organic layer was washed with brine (240 mL), waterand then dried over MgSO₄. After evaporation of the solvent, 4.3 g of awhite powder was obtained. The aqueous phase was acidified to pH 1.Ethyl acetate (250 mL) was added, and the layers were separated. Theorganic layer was washed with brine (240 mL), water and then dried overMgSO₄. After evaporation of the solvent, 8.22 g of a white powder wasobtained.

B) Methyl 4-hydroxy-3-iodobenzoate: A solution of3-iodo-4-Hydroxybenzoic acid (7.25 g, 27.4 mmol) and sulfuric acid (1.9ml, 36 mmol) in methanol is stirred at 55° C. for 6 hours. TLC(dichloromethane): 30% of starting material. The solution is stirred 12h at room temperature. TLC (dichloromethane): 10% of starting materialand stirred at 55° C. for 2 h. After cooling, ethyl acetate (200 mL) wasadded and the mixture was adjusted to pH 3 using sodium bicarbonate. Theorganic layer was washed two times with water and then dried over MgSO₄.Filtration and rotary evaporation at 40° C. afforded a white solid. Thesolid was triturated with hexane, filtered off and dried under reducedpressure. M=4.47 g. Yield: 59%.

C) 4-Iodo-3-(2-methyl-allyloxy)-benzoic acid methyl ester: To a solutionof methyl 3-hydroxy-4-iodobenzoate (1.5 g, 5.4 mmol) in anhydrous methylethyl ketone (60 mL) was added finely powdered Potassium carbonate (1.49g, 10.78 mmol) followed by 3-bromo-2-methyl-propene (0.81 mL, 1.1 g,8.15 mmol). The reaction mixture was heated at 70° C. for 4 h. Themixture was diluted filtrated, washed with water and dried over MgSO₄.Evaporation of the solvent and of the remaining bromopropene in vacuoafforded the requisite alkylated ester as a yellow oil. M: 1.4 g, Yield:78%.

NMR (CDCl₃, ¹H): 1.90 (3H, d, J=1.2 Hz), 194 (3H, s), 4.56 (2H, s), 5.06(1H, d, J=1.2 Hz), 5.25 (1H, d, J=1.2 Hz), 7.38 (1H, dd, J=8.1 Hz, J=1.8Hz), 7.44 (1H, d, J=1.8 Hz), 7.88 (1H, d, J=1.8 Hz)

D) 3-B enzyl-3-methyl-2,3-dihydro-benzofuran-6-carboxylic acid methylester: To a solution of 4-Iodo-3-(2-methyl-allyloxy)-benzoic acid methylester (455 mg, 1.37 mmol) in DMF (15 mL) were added Potassium carbonate(379 mg, 2.74 mmol), Tetrabutylammonium chloride (380 mg, 1.37 mmol),Palladium acetate (25.6 mg, 0.136 mmol) in DMF (5 mL) and Phenylboronicacid (200 mg, 1.64 mmol). The resulting mixture was stirred for 3 h at115° C., cooled to room temperature, filtered over silica, washed withwater, dried over MgSO₄ and concentrated. Column chromatography (silicagel, heptane/CH₂Cl₂ : 4/6) afforded 368 mg (95%) of the title compoundas a slightly brown oil which crystallize. Mp: 52° C.

NMR (CDCl₃, ¹H): 1.38 (3H, s), 2.86 (1H, d, J=14 Hz), 2.93 (1H, d, J=14Hz), 3.89 (3H, s), 4.12 (1H, d, J=8.7 Hz), 4.55 (1H, d, J=8.7 Hz),6.93-6.98 (3H, m), 7.22-7.24 (3H, m), 7.38 (1H, d, J=1.2 Hz), 7.59 (1H,dd, J1=7.5 Hz, J2=1.2 Hz).

E) 3-Benzyl-3-methyl-2,3-dihydro-benzofuran-6-carboxylic acid: A mixtureof 3-B enzyl-3-methyl-2, 3-dihydro-benzofuran-6-carboxylic acid methylester (300 mg, 1.06 mmol), sodium hydroxide (260 mg, 6.5 mmol), ethanol(10 ml) and water (1 ml) in tetrahydrofuran (10 ml), is stirred for 12 hat room temperature. The reaction medium is acidified by adding a 1.2 Mhydrochloric acid solution and extracted with ethyl acetate. The organicphase is washed with water, dried (Na₂SO₄), and concentrated in a rotaryevaporator. The product is obtained as a white solid (300 mg, 100%). Mp:165° C.

NMR (CDCl₃, ¹H): 1.39 (3H, s), 2.87 (1H, d, J=14 Hz), 2.93 (1H, d, J=14Hz), 4.14 (1H, d, J=8.7 Hz), 4.57 (1H, d, J=8.7 Hz), 6.96-7.00 (3H, m),7.22-7.25 (3H, m), 7.45 (1H, d, J=1.2 Hz), 7.65 (1H, dd, J1=7.8 Hz,J2=1.2 Hz).

F) 3-benzyl-3-methyl-2,3-dihydrobenzofuran-6-carboxylic acid-piperidineamide: To a stirred suspension of the 3-Benzyl-3-methyl-2,3-dihydro-benzofuran-6-carboxylic acid (80 mg, 0.3mmol) and piperidine (28 mg, 33 μL, 0.33 mmol) in dichloromethane (3 mL)and DMF (2 mL) were addedO-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU) (125 mg, 0.33 mmol) and then a solution ofN,N-diisopropylethylamine (58 mg, 78 μL, 0.45 mmol, mL) in DMF (1 mL).The reaction mixture was stirred at ambient temperature for 18 h. Thereaction medium is acidified by adding a 1.2 M hydrochloric acidsolution and extracted with ethyl acetate. The organic phase is washedwith water, dried (MgSO₄), and concentrated to give the amide which ispurified by flash chromatography (AcOEt/heptane: 4/6) to afford 50 mg ofa white solid (yield: 50%).

NMR (CDCl₃, ¹H): 1.36 (3H, s), 1.54-1.67 (6H, m), 2.85 (1H, d, J=13.2Hz), 2.90 (1H, d, J=13.2 Hz), 3.35 (2H, m), 3.68 (2H, m), 4.09 (1H, d,J=8.7 Hz), 4.53 (1H, d, J=8.7 Hz), 6.75 (1H, m), 6.88 (1H, dd, 31=7.5Hz, J2=1.2 Hz), 6.94 (1H, d, J=7.5 Hz), 7.00 (2H, m), 7.21-7.24 (3H, m).

Example II: Activation of the CB₂ receptor system reversesamyloid-induced memory deficiency

In this study, the inventors demonstrate that MDA7 exerts aneuroprotective effect by blunting neuroinflammatory processes that arepivotal for the development of Aβ-induced neurotoxicity. They found thatMDA7 can: (1) promote Aβ clearance, (2) prevent increases in levels ofglial fibrillary acidic protein (GFAP) in astrocytes and of CD11b inmicroglia (indicative of neuroinflammatory process activation) that aretypically seen in the hippocampus of Aβ-injected rats; (3) decrease theproduction of interleukin (IL)-1β; (4) ameliorate the Aβ-mediatedsuppression of glutamatergic transmission in the hippocampus ofAβ-injected rats; and (5) prevent cognitive impairment on spatial memoryperformance using the Morris water maze test in the Aβ-injected rats.Because this CB₂ agonist prevented Aβ-induced neuroinflammation and itsdownstream consequence—synaptic plasticity and cognitive impairment itwas hypothesized that the CB₂receptor functions in a negative-feedbackloop and that its activation can induce Aβ clearance, and bluntneuroinflammatory responses and cognitive impairment induced by Aβfibrils.

Material and methods

Animals and treatment protocol. All animal procedures were approved bythe Animal Care and Use Committee of Cleveland Clinic. Animals werehoused the Institutional Biological Rodent Unit on a 12/12 h light/darkcycle with water and food pellets available ad libitum. Adult maleSprague-Dawley (Charles River, Wilmington, Mass.) rats weighing 200-250g were used, and all experiments were performed during the light cycle.

Animals were divided into several groups (40-50 rats per group). TheAβ₁₋₄₀ group received bilateral intracerebral microinjection of Aβ₁₋₄₀fibrils once and saline intraperitoneally (i.p.) daily for 14 days. TheAβ₁₋₄₀+MDA7 group received bilateral intracerebral microinjection ofAβ₁₋₄₀ fibrils once and 15 mg/kg MDA7 i.p. daily for 14 days. Othercohorts received smaller doses of MDA7 (5 mg/kg or 1.5 mg/kg) i.p. dailyfor 14 days to determine the dose-response characteristics for MDA7. Inother groups, AM630 (a CB₂ antagonist) 5 mg/kg i.p. was administereddaily for 14 days either alone (Aβ₁₋₄₀+AM630 group) or 15 min prior toMDA7 (15 mg/kg i.p.) administration (Aβ₁₋₄₀+MDA7+AM630 group) to ratsinjected with β₁₋₄₀ fibrils. MDA7 group and control group receivedbilateral intracerebral microinjection of artificial cerebrospinal fluid(130 mM NaCl, 2.6 mM KCl, 4.3 mM MgCl₂ and 1.8 mM CaCl₂) once and 15mg/kg MDA7or saline i.p. daily for 14 days, respectively.Intraperitoneal administration of AM630, MDA7 or saline started on thesame day after intracerebral microinjection of Aβ₁₋₄₀. All drugs wereprepared coded. Decoding was performed at the end of all experiments.

Microinjection of amyloid fibrils into the hippocampal CA1 area. Ratswere anesthetized with sodium pentobarbital (45 mg/kg i.p.) andrestrained in a stereotaxic apparatus. Aβ₁₋₄₀ fibrils were formed asdescribed previously. Chacon, et al., Mol Psychiatry 9(10), 953-61(2004). Aβ₁₋₄₀ fibrils (10 μg/3 μl) or 3 μl of artificial cerebrospinalfluid were injected stereotaxically and bilaterally into eachhippocampus (coordinates; Bregma −3.5 mm anteroposterior, ±2.0 mmmediolateral, and −3.0 mm dorsoventral) using a 10 μl Hamilton syringewith a 27 G stainless steel needle at a rate of 0.5 μl/min. Thisexperimental model has been used for studying AD. Ahmed, et al.,Neuroscience 169(3), 1296-306 (2010).

Morris water maze test. A cohort of rats (n=10 per group) was tested 15days after surgery as described by Chacon et al. (ibid). Testing wasconducted in all groups at the same time of the day. The experimentalapparatus consisted of a circular pool (120 cm in diameter, 45 cm high).An invisible platform (15 cm in diameter, 35 cm high) was placed 1.5 cmbelow the surface of the water. The water temperature was kept at 24-28°C. The pool was located in a test room, and many clues external to themaze were visible from the pool, which could be used by the rats forspatial orientation. The position of the cues was kept constantthroughout the task. Each rat underwent four trials per day for fivedays. During each trial (memory acquisition), the rats were placedrandomly at one of four fixed starting points and were allowed to swimfor 120 s or until they escaped from the water by reaching the platform.The platform was located in the same position throughout the test in themiddle of one quadrant. The start location was moved to a differentquadrant in each trial so that no single start location was used inconsecutive trials. In each training session, the latency to escape tothe hidden platform was recorded. After the rats reached the platform,they were allowed to rest there for 20 seconds, and they then wereremoved from the pool. An inter-trial interval of at least 4 minutes wasused to ensure that each rat's performance was not impaired by fatigue.After completing four trials, the rats were removed from the pool, driedand returned to their home cage. On Day 6, all rats were subjected toone probe trial (memory retrieval) in which the platform was removed,and each animal had 60 s to search the pool for the platform. Allbehavioral testing was performed by a single experimenter who wasblinded to the different treatment groups. After the behavioral test,the rats were sacrificed, and the hippocampus tissues were collected forfurther study.

Hippocampal slice preparation. Brain slices containing hippocampus wereprepared. Brain slices used for electrophysiological recording wereobtained from all rats (n=10-12 rats per group) that had been subjectedto the Morris water maze test. All rats were anesthetized withpentobarbital and then euthanized by decapitation. The brain was quicklyremoved and cut on a Vibratome (Leica VT-1000S) in cold (4° C.)physiological saline to obtain coronal slices (300 μm thick) containingthe hippocampus. A single slice was submerged in a shallow recordingchamber and perfused with warm (35° C.) physiological saline (126 mMNaCl, 2.5 mM KCl, 1.2 mM NaH₂PO₄, 1.2 mM MgCl₂, 2.4 mM CaCl₂, 11 mMglucose, and 25 mM NaHCO₃, saturated with 95% O₂ and 5% CO₂, pH7.3-7.4). Slices were maintained at approximately 35° C. throughout therecording experiment.

Whole-cell patch clamp recordings in hippocampal slices. The hippocampalslices were allowed to recover for 1 h after which they were visualizedusing an upright microscope with infrared illumination. Whole-cellvoltage-clamp recordings from the CA1 area were taken using an Axopatch200B amplifier (Molecular Devices) with 2-4 MΩ glass electrodescontaining the following internal solution (mM): K-gluconate or cesiummethanesulfonate, 125; NaCl, 5; MgCl₂ 1; EGTA, 0.5; Mg-ATP, 2; Na₃GTP,0.1; HEPES, 10; pH 7.3; 290-300 mOsmol. A seal resistance of ≥2 GΩ andan access resistance of 15-20 MΩ were considered acceptable. The seriesresistance was optimally compensated by ≥70% and constantly monitoredthroughout the experiments. The membrane potential was held at −70 mV,unless otherwise stated, throughout the experiment. Schaffercollateralcommissural fibers were stimulated by ultra thin concentricbipolar electrodes (FHC Inc., Bowdoinham, Me.), and the stimulusintensity was adjusted to evoke about 35% maximal stimulation unlessspecified. Excitatory postsynaptic currents (EPSCs) were recorded in theCA1 area in the presence of GABA_(A) antagonist bicuculline (30 μM). Theevoked EPSCs were filtered at 2 kHz, digitized at 10 kHz, and acquiredand analyzed using Axograph X software. The amplitude of EPSCs wasmonitored for a baseline period of at least 15 min. If synaptictransmission was stable (<15% change in EPSC amplitude over 15 min),long-term potentiation (LTP) was induced by a single high-frequencyelectric stimuli train (100 Hz for 1 s). All electrophysiologicalexperiments were performed at room temperature (23±2° C.).

Immunostaining. Immunostaining of the hippocampal slices was performed.Bie et al., J Neurosci 30(16), 5617-28 (2010). A cohort of rats (n=10-12 per group) were deeply anesthetized with 60 mg/kg sodiumpentobarbital and perfused transcardially with 0.1 m PBS followed by 4%formalin. The brainstem was collected, postfixed in the same fixativefor 4 h, and then cryoprotected in 30% sucrose in PBS for 3 days. Serialsections (30 μm, 15-20/rat) containing the hippocampal CA1 area were cutfrom the fixed brain. The sections were incubated for at least 1 h in0.01 m PBS with 0.3% Triton X-100 plus 5% normal donkey serum. Primaryand secondary antibodies were diluted in 0.01 m PBS with 0.3% TritonX-100 plus 1% bovine serum. Sections were processed overnight at 4°C.for double-labeling immunofluorescence using rabbit antibodiesdirected against CB₂ (1:500, Thermo Scientific, Rockford, Ill.), mousemonoclonal antibody against the microglial marker CD11b (1:200, Abcam,Cambridge, Mass.), and glial fibrillary acidic protein (GFAP) antibody(1:400; Abcam, Cambridge, Mass.). Other sections were processedovernight at 4° C.for immunofluorescence using Aβ₁₋₄₀ (11A50-B10)monoclonal antibody (1 μg/ml PBS, Covance San Diego Calif.). Then, thesections were incubated with a mixture of FITC-(1:500, JacksonImmunoResearch, West Grove, Pa.) or Cy3-conjugated secondary antibodies(1:500, Jackson ImmunoResearch, West Grove, Pa.) for 1 h. Omission ofprimary or secondary antibodies resulted in no immunostaining. Three tosix sections from each rat were randomly selected and were analyzedusing Leica DMLB fluorescence microscope (Leica Microsystems WetzlarGmbH, Germany) by one investigator, who was blinded to the origin oftissue being examined. Images were quantified using MCID Core softwareversion 7.0 (InterFocus Imaging Ltd, Cambridge, England).

Protein extraction and immunoblotting. The hippocampal CA1 tissues fromthe rats in all groups (n=10-12 rats per group) were gently homogenizedin ice-cold lysis buffer containing 50 mM TrisCl, 150 mM NaCl, 0.02 mMNaN₂, 100 μm/ml phenylmethyl sulfonyl fluoride, 1 μm/ml aprotinin, 1%Triton X-100 and proteinase inhibitor cocktail. The lysates werecentrifuged at 14,000 rpm for 10 min at 4° C., and the supernatant wasused for SDS-polyacrylamide gel electrophoresis. Protein concentrationswere determined by using the Bio-Rad (Hercules, Calif.) protein assaykit.

The samples were treated with SDS sample buffer at 95° C.for 5 min,loaded on a 7.5% SDS-polyacrylamide gel, and blotted to a nitrocellulosemembrane. The blots were incubated overnight at 4° C.with a rabbitpolyclonal anti-CB₂ primary antibody (1:100; Santa Cruz Biotechnology,Inc., Santa Cruz, Calif.), monoclonal anti-CD11b antibody (1:1000;Abcam, Cambridge, Mass.), monoclonal anti-GFAP antibody (1:1000; Abcam,Cambridge, Mass.), goat polyclonal anti-IL-1β antibody (1:300; Abcam,Cambridge, Mass.) or monoclonal anti-β-actin antibody (1:250; Santa CruzBiotechnology, Inc.). The membranes were washed extensively withTris-buffered saline and incubated with horseradishperoxidase-conjugated anti-mouse and anti-rabbit IgG or anti-goat IgGantibody (1:10,000; Jackson ImmunoResearch Laboratories Inc., WestGrove, Pa.). The immunoreactivity was detected using enhancedchemiluminescence (ECL Advance Kit; Amersham Biosciences). The intensityof the bands was captured digitally and analyzed quantitatively withImageJ software. The immunoreactivity of CB2, CD11b, and GFAP wasnormalized to that of β-actin.

Compounds. Aβ peptide consisting of residues 1-40 of the human wild-typesequence (Aβ₁₋₄₀) was purchased from Bachem (Torrance, Calif., USA).AP-5 (D-2-amino-5-phosphonopentanoate), CNQX(6-cyano-2,3-dihydroxy-7-nitroquinoxaline), bicuculline, and otherchemicals (NaCl, KC1, NaH₂PO₄, MgCl₂, CaCl₂, glucose, NaHCO₃, EGTA,Mg-ATP, Na₃GTP, HEPES and Triton-X 100) were purchased from SigmaAldrich (St. Louis, Mo.) or Tocris (Ellisville, Mo.). AM630(6-Iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl(4-methoxyphenyl)methanone) was purchased from Tocris (Ellisville, Mo.).MDA7 (hCB1 Ki value >10,1000 nM; hCB₂ Ki value=422 nM; rCB₁ Kivalue=2565 nM; rCB₂ Ki value=238 nM; EC₅₀ at hCB₁=not active, EC₅₀ athCB₂=128 nM; ECso at rCB₁=not active; EC₅₀ at rCB₂=67 nM) wassynthesized as previously described. Diaz, et al., ChemMedChem 4(10),1615-29 (2009).

Data analysis and statistics. The behavioral data were analyzed usingrepeated measures ANOVA followed by post hoc Student-Newman-Keulsmultiple range test. Immunostaining and immunoblotting data wereanalyzed using one-way ANOVA followed by post hoc Student-Newman-Keulsmultiple range test for group means. The evoked EPSC and LTP data wereanalyzed using repeated measures ANOVA followed by post hocStudent-Newman-Keuls multiple range test. All analyses were performedusing the BMDP statistical package (Statistical Solutions, Saugus,Mass.). Results were expressed as mean and SEM, and they were consideredsignificant when P <0.05.

Results

Effect of activation of CB₂ on the behavioral test. Firstly, the effectof i.p. daily administration (for 14 days) of three different doses ofMDA7 (1.5 mg/kg, 5 mg/kg, or 15 mg/kg) on cognitive impairment inducedby Aβ₁₋₄₀ was determined. Repeated measures ANOVA identified asignificant dose-dependent memory enhancing effect of MDA7 in the Morriswater maze test (overall F_((4,45))=6.86, n=50, P=0.0002). Animalsinjected with Aβ₁₋₄₀ fibrils and treated with 15 mg/kg MDA7 i.p. for 14days had a significantly shorter escape latency in the Morris water mazetest than the animals injected with Aβ₁₋₄₀ fibrils and treated with i.p.saline for 14 days (FIG. 1A) at day 3 (P<0.05), day 4 (P<0.05), and day5 (P<0.05). Similar observation was noted during the probe trial, inthat the rats injected with Aβ₁₋₄₀ fibrils and treated with i.p. MDA7 15mg/kg for 14 days spent the longest time in the target quadrant (TQ orplatform quadrant) than animals injected with Aβ₁₋₄₀ and treated withi.p. saline for 14 days (P<0.05) (FIG. 1B). It was noted that the effectof 1.5 mg/kg MDA7 i.p. for 14 days did not result in any significantmemory enhancement following Aβ₁₋₄₀ fibrils administration, however, theeffect of the 5 mg/kg MDA7 dose was limited only to days 3 and 5 (FIG.1A). Based on these data, the dose of 15 mg/kg of MDA7 was selected forfurther studies.

Repeated measures ANOVA for the data reported in FIG. 1C identifiedsignificant differences among the six groups (overall F_((5,54))=8.10,n=60, P <0.0001). The rats in the Aβ₁₋₄₀ group had a significantlyextended escape latency in the Morris water maze test than those inAβ₁₋₄₀+MDA7 group or the control group (FIG. 1C) at day 3 (P<0.01), day4 (P<0.01), and day 5 (P<0.01). During the probe trial (FIG. 1D), ratsin the Aβ₁₋₄₀+MDA7 group spent significantly longer time in the targetquadrant than those in the Aβ₁₋₄₀ group (P<0.01). This indicated thatMDA7 treatment was able to prevent the cognitive impairment on spatialmemory performance induced by Aβ₁₋₄₀.

To confirm the CB₂-specific effect of MDA7, 5 mg/kg of AM630, a CB₂antagonist, was administered i.p. daily for 14 days either alone or 15min prior to MDA7 (15 mg/kg i.p.) administration to groups of ratsinjected with Aβ₁₋₄₀ fibrils. As shown in FIG. 1C, administration ofAM630 prior to MDA7 treatment reversed the effect of MDA7 on the escapelatency. Similar effect of AM630 was also noted in the probe test (FIG.1D). Meanwhile, rats received AM630 alone i.p. (in the Aβ₁₋₄₀+AM630group) did not show any improvement in spatial memory or probe testcompared to that in the Aβ₁₋₄₀ group (FIG. 1C and D). The differentbehavioral performances noted in this study were not attributable to thepresence of motor deficits because all groups of rats exhibited similarswimming speeds (data not shown). The results indicated the effect ofMDA7 on restoring cognition and memory was specifically mediated throughthe CB₂ receptor system.

MDA7 effectively ameliorates Aβ₁₋₄₀-induced glial activation in thehippocampal CA1 area. The accuracy of the microinjection washistologically verified afterwards. FIG. 2A shows an India ink-markedmicroinjection site in the hippocampal CA1 area demonstrating thepreciseness of the injection site.

Prior studies have found an association between AD and glial cellactivation. Akiyama et al., Neurobiol Aging 21(3), 383-421 (2000), Perryet al., Nat Rev Neurol 6(4) (2010). The possibility that MDA7'spreventive effect on Aβ₁₋₄₀-induced microglial activation was mediatedthrough modulation of glial activation was therefore considered.Increased expression of specific markers such as CD11b and GFAP havebeen associated with activation of microglia (Eng, J Neuroimmunol8(4-6), 203-14 (1985)) and astrocytes, respectively.

The impact of 15 mg/kg of MDA7 i.p. daily for 14 days on Aβ₁₋₄₀-inducedmicroglial and astrocyte activation was examined by comparing the levelsof immunofluorescence staining and immunoblotting intensity for CD11b(FIG. 2) and GFAP (FIG. 3) in the hippocampal CA1 area of rats in thevarious treatment groups. Glial activation is characterized by anincrease in the number and complexity of these cells (rounded cellbodies and thicker processes), resulting in an increase in both thenumber of labeled cells and the total integrated intensity of thelabeled cells (FIGS. 2B and 3A). When the hippocampal CA1 areas wereexamined in the rats after microinjection of Aβ₁₋₄₀ and treatment withi.p. saline for 14 days, increased CD11b and GFAP immunoreactivity,enlarged cell mass, and increased cell complexity were noted in themicroglia (FIGS. 2B and 2C, F_((3,76))=243.2, n=80 sections (3-5sections per animal from 5 animals per group), one-way ANOVA followed byStudent-Newman-Keuls multiple range test, P<0.01) and astrocytes (FIGS.3A and 3B, F_((3,76))=225.3, n=80 sections (3-5 sections per animal from5 animals per group), P<0.01), respectively, compared to the controlgroup.

The protein expression of CD11b (FIGS. 3D and 3E, n=6 rats per group,F_((1,10))=19.9, one-way ANOVA followed by Student-Newman-Keuls multiplerange test, P<0.01) and GFAP (FIGS. 3C and 3D, n=5 rats per group,F_((1,8))=32.7, P<0.01) was significantly higher in the rats injectedwith Aβ₁₋₄₀ and treated with i.p. saline for 14 days than in the controlgroup rats. These results confirmed that Aβ₁₋₄₀-induced braininflammation is characterized by the activation of microglia andastrocytes in the hippocampal CA1 area. The changes in CD11b and GFAPexpression were significantly attenuated in the Aβ-injected rats thatreceived 14 days of MDA7 i.p. treatment compared to the animals injectedwith Aβ₁₋₄₀ and treated with i.p. saline for 14 days (FIGS. 2D and 2E,n=6-7 rats per group, F_((1,11))=16.1, one-way ANOVA followed byStudent-Newman-Keuls multiple range test, P<0.01) (FIGS. 3C and 3D, n=5rats per group, F_((1,8))=46.2, P<0.01). Treatment with MDA7 alone hadno significant effect on the CD11B or GFAP expression compared to thecontrol rats.

MDA7 modulated Aβ₁₋₄₀-induced CB₂ receptor upregulation in thehippocampal CA1 area. Previous studies by the inventors established MDA7as a potent and selective CB₂ agonist. Naguib, et al., Br J Pharmacol155(7), 1104-16 (2008). Herein, the effect of Aβ₁₋₄₀ fibrils on theexpression of CB₂ receptor in the hippocampal CA1 area was examined. Itwas found that expression of CB₂ receptors was enhanced with Aβ₁₋₄₀administration (FIGS. 4A and B, F_((3,76))=48.45, n=80 sections (3-5sections per animal from 5 animals per group), one-way ANOVA followed byStudent-Newman-Keuls multiple range test, P<0.01 versus control group)and this expression is colocalized with the reactive microglia.Administration of 15 mg/kg MDA7 i.p. for 14 days to rats injected withAβ₁₋₄₀ resulted in reduced CB₂ expression (P<0.01 versus rats injectedwith Aβ₁₋₄₀ and treated with i.p. saline for 14 days).

The immunoblotting studies revealed that CB₂ receptor expression in therats that received microinjection of Aβ₁₋₄₀ and treatment with i.p.saline for 14 days (FIGS. 4C and 4D, n=5, F_((1,8))=10.3, one-way ANOVAfollowed by Student-Newman-Keuls multiple range test, P<0.05) wasgreater than that in the control rats. Furthermore, treatment with 15mg/kg of MDA7 i.p. daily for 14 days significantly reversed this upsurgeof CB₂ receptor expression induced by Aβ₁₋₄₀ fibrils (FIGS. 4C and 4D,n=5, F_((1,8))=19.1, one-way ANOVA followed by Student-Newman-Keulsmultiple range test, P<0.01). MDA7 did have any effect on the CB₂receptor expression in the control rats. These results represent anadaptation of brain CB₂ receptor induced by Aβ₁₋₄₀ fibrils and itsmodulation by the CB₂ agonist MDA7.

MDA7 attenuates IL-1β protein expression induced by Aβ₁₋₄₀ in thehippocampal CA1 area. To better understand the basis for MDA7'sinterference with Aβ-induced neuroinflammation, the impact of MDA7treatment (15 mg/kg i.p. daily for 14 days) on proinflammatory cytokineIL-1 r3 production was examined in the hippocampal CA1 area. In thebrain, IL-1β is mostly synthesized and secreted from the activatedmicroglia and astrocytes (Kettenmann et al., Physiol Rev 91(2), 461-553(2011)), and it is implicated in the development of brain inflammation(Gabay et al., Nat Rev Rheumatol 6(4), 232-41 (2010)) andamyloid-induced memory deficiency (Schmid et al., Hippocampus 19(7),670-6 (2009)). In the present study, in animals injected with Aβ₁₋₄₀ andtreated with i.p. saline for 14 days showed significantly higherexpression of the IL-1β protein in the hippocampal CA1 tissue (FIG. 5,n=5, F_((1,8))=63.3, one-way ANOVA followed by Student-Newman-Keulsmultiple range test, P<0.01) than the control rats (n=5). Meanwhile,MDA7 treatment for 14 days in the rats injected with Aβ₁₋₄₀significantly attenuated the upsurge of IL-1β induced by microinjectionof amyloid fibrils (FIG. 5, n=5, F_((1,8))=34.6, P<0.01). The resultsindicate that the levels of IL-1β from activated glial cells weresignificantly increased in the hippocampal CA1 area after microinjectionof Aβ₁₋₄₀, and these increases were blunted by MDA7 treatment for 14days.

MDA7 effectively promotes the clearance of Aβ₁₋₄₀ injected in thehippocampal CA1 area. In vitro studies (Tolon et al., Brain Res 1283,148-54 (2009)) have shown that the CB₂ activation induces removal ofβ-amyloid and this effect was blocked by CB₂ antagonists. The inventorsthus hoped that MDA7 would enhance clearance mechanisms of the injectedAβ₁₋₄₀ in rats. MDA7 treatment for 14 days (15 mg/kg per day) in therats injected with Aβ₁₋₄₀ induced removal of Aβ₁₋₄₀ (FIG. 6,F_((3,76))=707.07, n=80 sections (3-5 sections per animal from 5 animalsper group), one-way ANOVA followed by Student-Newman-Keuls multiplerange test, P<0.01) and this effect was abrogated by prioradministration of 5 mg/kg AM630 i.p. for 14 days. The inventors alsoobserved that administration of 5 mg/kg AM630 i.p. alone has no effecton the clearance of the injected Aβ₁₋₄₀. The results indicate thatMDA7-mediated cognitive improvements are correlated with the enhancedclearance of Aβ peptide.

MDA7 effectively ameliorates Aβ₁₋₄₀-impaired glutamatergic transmissionin the hippocampal CA1 area. Then, the input (stimulationintensity)output (current amplitude) responses of the evoked EPSCs inthe CA1 neurons were compared among all groups. As shown in FIGS. 7A and7B, the amplitudes of evoked EPSCs were significantly less in the Aβ₁₋₄₀group than that in the control group at all three stimulus intensities,indicating attenuated basal glutamatergic strength in the CA1 neurons.However, the amplitude of evoked EPSCs in the CA1 neurons from the ratsinjected with Aβ₁₋₄₀ in rats was restored by 15 mg/kg MDA7 i.p.treatment for 14 days in the Aβ₁₋₄₀+MDA7 group at all three stimulusintensities (FIG. 7A and B). The basal glutamatergic strength in thehippocampal slices was not different between the control and MDA7groups. These results indicated that administration of MDA7significantly ameliorated the impaired basal glutamatergic strengthinduced by microinjection of APβ₁₋₄₀ fibrils into the hippocampus.

In the hippocampus slice of the saline-injected rats, high frequencyelectric stimuli on the Schaffer collateralcommissural fibers inducedsignificant synaptic potentiation in CA1 neurons that lasted more thanone hour. The average LTPs were 210.5±18.6% and 224.7±2.2% at 30 min and60 min after induction, respectively (n=12, repeated measures ANOVAfollowed by Student-Newman-Keuls multiple range test, P<0.01 whencompared with baseline, FIGS. 7C and 7D). Meanwhile, in the hippocampusslice of the Aβ₁₋ ₄₀-injected rats, the intensity of LTP induced by highfrequency electric stimuli was significantly attenuated. The averageLTPs were 107.1±14.0% and 110.4±15.6% at 30 min and 60 min afterinduction, respectively (n=10, repeated measures ANOVA followed byStudent-Newman-Keuls multiple range test, P<0.01 when compared with thatin the saline-injected rats, FIGS. 7C and 7D). However, in thehippocampus slices from the Aβ+MDA7 group, the average LTPs were214.5±23.6% and 195.7±21.8% at 30 min and 60 min after induction,respectively (n=9, repeated measures ANOVA followed byStudent-Newman-Keuls multiple range test, P<0.01 when compared with thatin the Aβ₁₋₄₀-injected rats, FIGS. 7C and 7D). Note that theadministration of MDA7 alone failed to significantly affect theinduction of LTP in the hippocampal slices of the control rats. Theseresults indicated that administration of MDA7 significantly amelioratedthe impaired hippocampal synaptic plasticity induced by microinjectionof Aβ₁₋₄₀ fibrils.

Discussion

The results reveal a promising potential role for the CB₂ agonist MDA7in (i) promoting Aβ clearance; (ii) ameliorating Aβ-induced glialactivation and cytokine production; and (iii) restoring of synapticplasticity, cognition and memory. The effects of MDA7 were abrogated byprior administration of a CB₂ antagonist AM630. The administration ofAM630 alone did not result in any beneficial effect on Aβ-relatedpathology. The present in vivo study confirmed that microinjection ofAβ₁₋₄₀ fibrils significantly (P<0.01) induced the activation ofastrocytes and microglia and upregulation of CB₂ receptors in thehippocampal CA1 area and that MDA7 treatment reversed this process. Thefinding that microinjection of Aβ₁₋₄₀ in rats treated with i.p. salinewas associated with increased CB₂ expression in the hippocampal CA1 area(FIG. 4) whereas treatment with the CB₂ agonist MDA7 ameliorated theeffects of amyloid fibrils raises the possibility that the CB₂ receptoracts as a negative feedback regulator and its activation by MDA7 canserve to limit the extent of the neuroinflammatory response and thesubsequent development of Aβ-induced neurotoxicity.

In the present study, microglial (CD11b) and astrocyte (FGAP) activationmarkers (measured on day 15) were significantly increased (P<0.01) inthe hippocampal CA1 area after microinjection of Aβ₁₋₄₀ in rats treatedwith i.p. saline compared to controls (FIGS. 2 and 3). Aβ aggregates arepotent neurotoxins and microglial activators (Cameron and Landreth,2010). The results are consistent with previously published reports inwhich intracerebral microinjection of Aβ₁₋₄₀ in the hippocampus, frontalcortex, or intracerebroventricular administration of Aβ₂₅₋₃₅ in that thecentral administration of Aβ, is associated with microglia (Ramirez etal., J Neurosci 25(8), 1904-13 (2005)) and astrocyte activation. Thisneuroinflammatory process appears to contribute to and/or reflectneuronal dysfunction in the brain and is found to be inverselycorrelated with the cognitive function in AD (Edison et al., NeurobiolDis 32(3), 412-9 (2008). Furthermore, in brain tissues from patientswith AD, microglia and astrocyte activation, evidenced by cellularhypertrophy, increases in the expression of GFAP, astroglial S100B, andCD11b proteins, are routinely observed. Although micromolarconcentrations of Aβ can induce direct toxicity to neurons,concentrations at nanomolar levels can induce neuronal loss through amicroglia-mediated mechanism. It seems that the deposition of Aβrepresents important trigger factors in glial activation (possibly viainteraction with microglial surface receptors(El Khoury et al., Nature382(6593), 716-9 (1996)) leading to an inflammatory reaction in thebrain. Meda et al., Neurobiol Aging 22(6), 885-93 (2001). Although ithas been reported that the administration of SR144525, a CB₂ antagonist,could have a role in decreasing Aβ₁₋₄₀-induced astrocyte activation, ourdata indicate that administration of 5 mg/kg AM630 (a CB₂ antagonist)i.p. for 14 days to rats injected with Aβ₁₋₄₀ failed to improvecognitive functions or induce Aβ clearance.

The results showed increased levels of IL-1β after bilateralmicroinjection of Aβ₁₋₄₀ in the hippocampus, and this increase wasprevented by MDA7 treatment. CB₂ receptor activation has been shown todecrease the production of proinflammatory molecules in vitro in ratmicroglial cells, human microglial cells (Stella, Glia 48(4), 267-77(2004)), and human astrocytes, and in animal models of perinatalhypoxiaischemia, Huntington's disease, and paclitaxel-inducedneuroinflammation. Naguib et al., Anesth Analg, 114(5):1104-20 (2012).IL-1β is synthesized and released from the activated microglia andastrocytes in the brain and is actively involved in the development ofamyloid-induced brain inflammation, impaired glutamatergic transmissionand memory deficiency. Intracerebroventricular administration of IL-1βsignificantly induced memory deficiency evidenced by impairedperformance in several memory tasks. In the primary culture ofhippocampal neurons, IL-1β (but not IL-10 or tumour necrosis factor-α)significantly downregulated the surface expression and Ser831phosphorylation of the AMPA receptor subunit GluR1, which plays animportant role in synaptic plasticity. Antagonism of IL-1 receptor inthe brain alleviated the impaired synaptic plasticity induced by Aβ.These previous reports established IL-1β, released from the activatedmicroglia and astrocytes, as an important factor for the pathogenesisand development of Alzheimer's disease.

In the present study, an increased expression of CB₂ receptors in thehippocampal CA1 tissue in the rats was noted following microinjection ofAβ₁₋₄₀ fibrils. MDA7 appears to be effective in suppressing microglialand astrocyte activities and IL-1β production through activation of theCB₂ receptor system. Previous studies have demonstrated that CB₂receptors expression in glia-associated plaques was increased in thepostmortem AD brains. Ramirez et al., J Neurosci 25(8), 1904-13 (2005).Intracerebroventricular administration of a non-selective cannabinoidreceptor agonist WIN 55,212-2 was effective in preventingAβ₂₅₋₃₅-induced microglial activity. Different cannabinoids (WIN55,212-2, HU-210, and JWH-113) blocked Aβ₁₋₄₀-induced activation ofcultured microglial cells. Ramirez et al., Ibid. In primary microglialculture, Aβ₁₋₄₂-induced activation of CB₂ receptors and administrationof JWH-015 (a CB₂ agonist) markedly suppressed microglial cytokines andnitric oxide production and attenuated CD40-mediated inhibition ofmicroglial phagocytosis of Aβ₁₋₄₂. Ehrhart, et al., J Neuroinflammation2, 29 (2005).

In vitro activation of CB₂ receptor facilitated the removal of native Aβfrom human frozen tissue sections as well as removal of syntheticpathogenic peptide by a human macrophage cell line. Tolon et al., BrainRes 1283, 148-54 (2009). The data showed that activating CB₂ receptorsystem by MDA7 promoted Aβ₁₋₄₀ clearance (FIG. 6), possibly by restoringmicroglial phagocytic function. Microglia play an important role inpromoting the clearance and phagocytosis of Aβ and there is an inverserelationship between cytokine production and Aβ clearance. However, asthe AD progresses, microglia continue to produce proinflammatorycytokines, but lose their Aβ-clearing capabilities. Hickman et al., JNeurosci 28(33), 8354-60 (2008). Hence, the upregulation of microglialCB₂ receptor in the Alzheimer brain potentially positioned the CB₂receptor as an endogenous protective mechanism to limit theneuroinflammation and pathological development in this disease. Thebeneficial effects of CB₂ agonist may not only rely on their ability toblock Aβ-induced microglial activation, but also to restore microglialabilities to remove Aβ deposits.

The behavioral deficits that were observed following hippocampalmicroinjection of Aβ₁₋₄₀ fibrils (FIG. 1B) correlated well withalteration in the hippocampal glutamatergic transmission (FIG. 7). Thepresent study, for the first time, demonstrated that systemicadministration of CB₂ agonist significantly ameliorated the impairedbasal glutamatergic strength and electric stimuli-induced synapticplasticity in the hippocampal CA1 area and the memory deficiency inducedby the local microinjection of Aβ₁₋₄₀ fibrils. It was previouslyreported that microinjection of amyloid fragments significantlyattenuated basal glutamatergic strength in the hippocampus of rodents.Intracerebroventricular injection of Aβ-containing aqueous extracts ofAlzheimer's disease brain significantly inhibited high-frequencyelectric stimuli-induced LTP—a form of synaptic plasticity—in the rathippocampus. Hippocampal LTP is used as a correlate for learning andmemory and has emerged as a valuable model for studying mechanismsinvolved in cognitive deficits related to AD. Administration ofexogenous Aβ into the hippocampus significantly lowered the performancein the memory tasks, such as the water maze, in rats. Consistently, inthe present study, an impaired electric stimuli-induced LTP and basalglutamatergic strength and extended escape time in the Morris water mazetest were observed in the rats 15 days after the microinjection ofAβ₁₋₄₀ fibrils in the rats treated with i.p. saline for 14 days.Interestingly, the impaired synaptic plasticity and memory deficiencywere normalized by the systemic administration of 15 mg/kg MDA7 for 14days.

In conclusion, activation of central microglial CB₂ receptors by MDA7(i) promoted AP clearance, (ii) ameliorated Aβ-induced glial activationand production of IL-1β, and (iii) restored synaptic plasticity,cognition and memory. The presence of CB₂ receptors in microglia in thehuman AD brain indicate that the use of CB₂ receptor agonists such asMDA7 may represent a novel therapeutic method for the treatment of AD.

The complete disclosure of all patents, patent applications, andpublications, and electronically available materials cited herein areincorporated by reference. The foregoing detailed description andexamples have been given for clarity of understanding only. Nounnecessary limitations are to be understood therefrom. In particular,while theories may be presented describing possible mechanisms throughwith the compounds of the invention are effective, the inventors are notbound by theories described herein. The invention is not limited to theexact details shown and described, for variations obvious to one skilledin the art will be included within the invention defined by the claims.

What is claimed is:
 1. A method of treating Parkinson's disease in asubject by administering a pharmaceutically effective amount of a CB₂receptor agonist to the subject, wherein the CB2 receptor agonist is acompound according to formula II


2. The compound of claim 1, wherein the compound is the S enantiomer. 3.The method of claim 1, wherein the CB2 receptor agonist is administeredparenterally.
 4. A method of activating CB2 receptors in CNS microgliaof a subject having Parkinson's disease by administering apharmaceutically effective amount of a CB2 receptor agonist to thesubject, wherein the CB2 receptor agonist is a compound according toformula II


5. The method of claim 4, wherein the subject has elevated levels ofamyloid-β peptide in the brain.
 6. The method of claim 4, whereinactivating the CB2 receptors increases one or more of synapticplasticity, cognition, or memory of the subject.
 7. The method of claim4, wherein activating the CB2 receptors decreases production of IL-1β bythe microglia.
 8. The method of claim 4 wherein activating the CB2receptors increases glutamatergic neurotransmission in the subject.